Plant pathogen inhibitor combinations and methods of use

ABSTRACT

Combinations, compositions and methods of use for modulating plant pathogen infection using plant extracts containing anthraquinone derivatives which induce resistance to plant phytopathogens and an antimicrobial agent, a biological control agent and/or a surfactant having fungicidal activity

TECHNICAL FIELD

Disclosed herein are combinations, compositions and methods of use formodulating plant pathogen infection using plant extracts containinganthraquinone derivatives which induce resistance to plantphytopathogens and an antimicrobial agent, a biological control agentand/or a surfactant having fungicidal activity.

BACKGROUND Plant Resistance to Plant Pathogens

Plants have evolved highly effective mechanisms for resistance todisease caused by infectious agents, such as bacteria, fungi andviruses. This resistance can be caused by several mechanisms, the bestknown of which are the systemic acquired resistance (SAR; Ross, 1961;Durrant and Dong, 2004) and induced systemic resistance (ISR; van Loonet al., 1998). In the most simple case, the inducer is the plantpathogen itself, in other cases, the inducer can be either a chemicalcompound (salicylic acid, benzo(1,2,3)thiadiazole-7-carbothioic acidS-methyl ester also known as BTH) or physical impact such as water orheat stress (Walters et al., 2005). It appears that induced systemicresistance depends upon a gradual expression and persistence of a lowlevel of metabolic perturbation. Unlike elicitors of phytoalexinaccumulation, which elicit at the site of application and may beresponsible for localized protection, inducers of systemic resistancesensitize the plant as a whole to respond rapidly after infections.These responses include phytoalexin accumulation, lignification andenhanced activities of chitinase and glucanase.

Extract from giant knotweed (Reynoutria sachalinensis) sold as MILSANA®and REGALIA® by Marrone Bio Innovations, Inc.) provides control ofpowdery mildew and other plant diseases on cucurbits and other cropsmainly by inducing an accumulation of fungitoxic phenolic compounds inthe plant (Daayf et al., 1995; Wurms et al. 1999; Schmitt, 2002).Recently, formulated giant knotweed extract has also shown greatefficiency in inducing resistance in various crops and plant pathogensincluding wheat powdery mildew (Vechet et al., 2009). Besides the ISRmode of action, the formulated R. sachalinensis extract has recentlyalso been shown to have a direct fungistatic effect against wheatpowdery mildew (Blumeria graminis f. sp. tritici; Randoux et al., 2008).

Fungicide Resistance

Fungicide resistance is a common phenomenon in pests including plantpathogens. When a fungicide, especially those with single-site mode ofaction, is frequently used, the targeted pathogen can adapt to thefungicide due to high selection pressure. It is estimated that pests candevelop resistance to pesticides within 5-50 generations (May, 1985).Most plant pathogens fit in this range in one growth season and thus candevelop fungicide resistance quickly. For example, it only took one yearfor benomyl lost efficacy for control of cucurbit powdery mildew afterits first registration for commercial use (McGrath, 2001).

Quinone outside inhibitors (also known as QoI fungicides orstrobilurins) has been widely used to control agriculturally importantfungal pathogens since their introduction in 1996. Strobilurins blockthe respiration pathway by inhibiting the cytochrome bc1 complex inmitochondria, thereby blocking the electron transfer process in therespiration chain and causing an energy deficiency due to lack ofadenosine triphosphate (ATP) (Bartlett et al., 2002). Strobilurins andother fungicides with a single-site mode of action such as demethylationinhibitors (DMI) are prone to resistance development among plantpathogens. To date, several plant pathogenic fungi have developed fieldresistance to strobilurins (Tuttle McGrath, 2003; Fraaije et al., 2003)and DMI fungicides (Schnabel et al., 2004), and considerable effort hasbeen made worldwide to develop appropriate resistance managementstrategies with detailed recommendations of how to combine fungicidesand other antifungal compounds in programs and rotations to minimize therisk of resistance development (Tuttle McGrath, 2006; Wyenandt et al.,2009).

Methods to Control Fungicide Resistance

The most common strategy to manage fungicide resistance is to usesite-specific fungicides that are prone to resistance development in acombination (pre-mix or tank mix). Besides resistance management, tankmixes also offer a compensatory mechanism in case of a failure of onefungicide as well as a way to reduce the dose to reduce selectionpressure on pathogens (van den Bosch and Gilligan, 2008). In some cases,the combination of single and multisite fungicides in a tank mix or inrotation can provide additive or even synergistic interactions (Gisi,1996). Holb and Schnabel (2008) were able to show improved control ofbrown rot (Monilinia fructicola) in a field study with a tank mix of aDMI fungicide and elemental sulfur, and Reuveni (2001) demonstrated thebenefits of using strobilurins and polyoxin B fungicides in combinationwith sulfur to control powdery mildew in nectarines.

Plant defense inducers such as the extract of R. sachalinensis have beentested in tank mixes and rotations with other SAR/ISR products as wellas with biocontrol agents (BCA) (Hafez et al., 1999; Belanger andBenyagoub, 1997; Schmitt et al., 2002; Schmitt and Seddon, 2005; Bardinet al., 2008). The purpose of these studies has mainly been todemonstrate the compatibility of different types of plant extracts withbiocontrol agents. Konstatinidou-Doltsinis et al. (2007) tested the R.sachalinensis product in a rotation with Pseudozyma flocculosa productagainst powdery mildew on grapes, and found that alternated applicationof both products improved the efficacy of R. sachalinensis. In the samestudy, alternation of sulfur and R. sachalinensis in a rotation did nothave a beneficial effect. Belanger and Benyagoub (1997) found that ayeast-like fungus, Pseudozyma flocculosa, was compatible with R.sachalinensis when used against cucumber powdery mildew in a greenhouse.Similarly, Bokshi et al. (2008) evaluated the combined effect of anacquired systemic resistance activator benzothiadiazole and MILSANA®against cucumber powdery mildew, and found that MILSANA® used in arotation with benzothiadiazole provided an effective control measureagainst powdery mildew in the field. However, based on the diseaseseverity and yield data collected, it was not possible to determinewhether the positive effect was additive or synergistic.

Pesticide synergism has been defined as “the simultaneous action of twoor more compounds in which the total response of an organism to thepesticide combination is greater than the sum of the individualcomponents” (Nash, 1981). Hence, when fungicides interactsynergistically, a high level of disease control is achieved with lessthan label rates of each individual fungicide. Usually, the best effectis achieved with combinations of fungicides with different modes ofaction (MOA), but synergy has also been demonstrated in combined use ofproducts with similar mode of action (De Waard, 1996). Fungicidesynergism has been demonstrated mostly in laboratory studies (Samouchaand Cohen, 1984; Gisi, 1996) but in some cases (Karaogladinis andKaradimos, 2006; Burpee and Latin, 2008) synergism has also been foundin the field studies. Additionally, synergism of antifungal compoundsother than fungicides (bicarbonates and refined petroleum distillate)has been demonstrated against rose powdery mildew and black spot (Horstet al., 1992).

SUMMARY

Disclosed and claimed is a combination comprising: (a) an extractderived from a plant, wherein said extract contains one or moreanthraquinone derivatives which induce plant resistance tophytopathogens (also referred to as “plant pathogens”) and (b) one ormore anti-phytopathogenic agents selected from the group consisting of:(i) a non-benzodiathiazole, non-Vitamin E, non-organophosphorusanti-microbial agent, which lacks or in other words does not containnon-elemental, non-wettable sulfur, (ii) a surfactant having fungicidalactivity and (iii) a non-Bacillus, non-Pseudomonas, non-Brevabacillus,non-Lecanicillium, non-Ampelomyces, non-Phoma, non-Pseudozyma biologicalcontrol agent (e.g., an agent derived from Streptomyces sp.,Burkholderia sp., Trichoderma sp., Gliocladium sp. or a natural oil oroil-product having fungicidal and/or insecticidal activity).

In a specific embodiment, the combination comprises: (a) an extractderived from the family Polygonaceae and (b) a non-benzodiathiazole,non-Vitamin E, non-organophosphorus anti-fungal and/or antibacterialagent, which lacks or does not contain non-elemental or non-wettablesulfur.

In one specific embodiment, the combination comprises (a) an extractderived from the family Polygonaceae (e.g. Reynoutria sachalinensis) and(b) a single site fungicide and/or multi-site fungicide which mayinclude but is not limited to myclobutanil, quinoxyfen, azoxystrobin,acibenzolar-S-methyl, mefenoxam, triflumizole, fludioxonil,propiconazole.

In another specific embodiment, the combination comprises (a) an extractderived from the family Polygonaceae (e.g. Reynoutria sachalinensis) and(b) a natural oil or oil-product having fungicidal and/or insecticidalactivity.

In yet another particular embodiment, the combinations are compositions,particularly compositions for use in modulating phytopathogenic orfungal infection. The invention is further directed to the use of theextract and anti-phytopathogenic agents in formulating thesecompositions.

The invention is additionally directed to a synergistic combination foruse in modulating phytopathogenic infection comprising (a) an extractderived from a plant, wherein said plant contains anthraquinonederivatives that induce plant resistance to phytopathogens and (b) anon-Vitamin E, non-organophosphorus antimicrobial agent (e.g.,anti-fungal and/or antibacterial agent), which lacks or does not containnon-elemental or non-wettable sulfur. In a particular embodiment, theantimicrobial agent is a benzodiathiazole (e.g., acibenzolar-S-methyl),a triazole (e.g., propiconazole) or a strobilurin (e.g., azoxystrobin).

The above mentioned combinations may also be formulated intocompositions.

The invention is further directed to a method for modulatingphytopathogenic infection in a plant comprising applying to the plantand/or seeds thereof and/or substrate used for growing said plant anamount of the combinations of the present invention set forth aboveeffective to modulate said phytopathogenic infection.

In a particular embodiment, the invention is directed to a method formodulating fungal and/or bacterial infection in a plant comprisingapplying to the plant and/or seeds thereof and/or substrate used forgrowing said plant an amount of the combinations of the presentinvention effective to modulate said fungal and/or bacterial infection.

The extract and said anti-phytopathogenic agents (e.g., anti-fungaland/or antibacterial agents) may be administered sequentially,concurrently or in combination intermittently. As defined herein“phytopathogenic infection” means infection of a plant by plantpathogenic bacteria, fungi, insects, nematodes and/or mollusks.

The invention is further directed to a method for decreasing theresistance of a phytopathogen (e.g., fungus and/or bacteria) to (i) anon-Vitamin E, non-organophosphorus anti-microbial agent, which lackedor in other words did not contain non-elemental or non-wettable sulfur,(ii) a surfactant having fungicidal activity and/or (iii) anon-Bacillus, non-Pseudomonas, non-Brevabacillus, non-Lecanicillium,non-Ampelomyces, non-Phoma, non-Pseudozyma biological control agent(e.g., an agent derived from Streptomyces sp., Burkholderia sp.,Trichoderma sp., Gliocladium sp. or a natural oil or oil-based producthaving fungicidal and/or insecticidal activity) comprising applying to aplant in need thereof an amount of the combinations of the presentinvention effective to decrease the emergence of said resistance. In aparticular embodiment, the invention is directed to a method fordecreasing the resistance of a fungus and/or bacteria to a non-elementalor non-wettable, sulfur, non-benzodiathiazole, non-Vitamin E,non-organophosphorus anti-fungal and/or antibacterial agent. In yetanother particular embodiment, the invention is directed to a method fordecreasing resistance of a fungus and/or bacteria to a natural oil oroil-based product having fungicidal and/or insecticidal activity usingthe combination of the present invention. This may be accomplished bydecreasing the frequency or rate of emergence. The resistance of aphytopathogen to the above-mentioned anti-pathogenic agents may bereduced by at least 50%.

The invention is further directed to the use of (a) an extract derivedfrom a plant, wherein said extract contains anthraquinone derivativeswhich induce plant resistance to phytopathogens, and (b) one or moreanti-phytopathogenic agents selected from the group consisting of: (i) anon-benzodiathiazole, non-Vitamin E, non-organophosphorus anti-microbialagent which lacks nonelemental, non-wettable sulfur, (ii) a surfactanthaving fungicidal activity and (iv) a non-Bacillus, non-Pseudomonas,non-Brevabacillus, non-Lecanicillium, non-Ampelomyces, non-Phoma,non-Pseudozyma biological control agent for the manufacture of acomposition for modulating phytopathogenic infection as well as the useof (a) an extract derived from a plant, wherein said plant containsanthraquinone derivatives that induce plant resistance to phytopathogensand (b) a benzodiathiazole anti-microbial agent for the manufacture of asynergistic composition for modulating phytopathogenic infection.

The invention is further directed to a kit comprising (a) an extractderived from the family Polygonaceae and (b) one or moreanti-phytopathogenic agents selected from the group consisting of: (i) anon-benzodiathiazole, non-Vitamin E, non-organophosphorus,anti-microbial agent, which lacks a non-elemental or non-wettablesulfur, (ii) a surfactant having fungicidal activity and (iii) anon-Bacillus, non-Pseudomonas, non-Brevabacillus, non-Lecanicillium,non-Ampelomyces, on-Phoma, non-Pseudozyma biological control agent. Thiskit may further comprise packaging instructions.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. Further, although thisinvention has been described with reference to specific embodiments, thedetails thereof are not to be construed as limiting, as it is obviousthat one can use various equivalents, changes and modifications andstill are within the scope of the present invention.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise. For example, “a fungus” alsoencompasses “fungi”.

As defined herein, the term “modulate” is used to mean to alter theamount of phytopathogenic infection or rate of spread of phytopathogenicinfection.

Extracts

The plant extracts used in the combinations, compositions and methods ofthe present invention contain anthraquinone derivatives as biochemicalagricultural products for use against plant pests, particularly plantphytopathogens such as plant pathogenic bacteria, fungi, insects,nematodes and/or as a molluscicide. “Contain” also encompasses extractsthat produce said anthraquinone derivatives. In a particular embodiment,the anthraquinone derivative(s) of the present invention which is usedin compositions and methods of the present invention is (are) the majoractive ingredients or one of the major active ingredients.

Anthraquinone derivatives include but are not limited to physcion,emodin, chrysophanol, ventiloquinone, emodin glycoside, chrysophanolglycoside, physcion glycoside, 3,4-dihydroxy-1-methoxyanthraquinone-2-corboxaldehyde, damnacanthal. These derivatives share asimilar structure as follows:

Where R1, R2, R3, R4, R5, R6, R7 and R8 are hydrogen, hydroxyl,hydroxylalkyl, halogen, carboxyl, alkyl, alkyoxyl, alkenyl, alkenyloxyl,alkynyl, alkynyloxyl, heterocyclyl, aromatic, or aryl group, sugars suchas glucose.

In a particular embodiment, the invention is directed to anthraquinonederivatives that are contained in extracts derived from plant familiesincluding but not limited to Polygonaceae, Rhamnaceae, Fabaceae,Asphodelaceae, and Rubiaceae so on. These compounds can be from any partof plants such as leaf, stem, bark, root and fruits. Plant materials canbe wet and dry, but preferably dry plant materials. To meet thebiochemical agricultural products, solvents and processes that are usedin the extraction and purification must meet the requirements ofNational Organic Program (NOP) [http://www.ams.usda.gov/AMSv1.0/nop,].

In a more particular embodiment, the plant extract is derived from amember of the Polygonaceae family. As defined herein, “derived from”means directly isolated or obtained from a particular source oralternatively having identifying characteristics of a substance ororganism isolated or obtained from a particular source. In a particularembodiment, extract in said combination contains at anthraquinonederivatives physcion and optionally emodin. Members of the Polygonaceaefamily include but are not limited to Acetosella, Antigonon,Aristocapsa, Bilderdykia, Brunnichia, Centrostegia, Chorizanthe,Coccoloba, Coccolobis, Coccolobo, Corculum, Dedeckera, Delopyrum,Dentoceras, Dodecahema, Emex, Eriogonum, Fafopyrum, Fagopyrum, Fallopia,Gilmania, Goodmania, Harfordia, Hollisteria, Koenigia, Lastarriaea,Mucronea, Muehlenbeckia, Nemacaulis, Oxyria, Oxytheca, Perscarioa,Persicaria, Pleuropterus, Podopterus, Polygonella, Polygonum,Pterostegia, Rheum, Rumex, Ruprechtia, Stenogonum, Systenotheca,Thysanella, Tovara, Tracaulon, Triplaris and even more particularembodiment, the extract may be derived from a Reynoutria (alternatelyreferred to as Fallopia) sp. or Rheum species. In a most particularembodiment, the extract is derived from Reynoutria sachalinensis.

Anti-Phytopathogenic Agents

The formulated extract (such as products marketed under trade namesREGALIA® and MILSANA) can then be used in combination with otheranti-phytopathogenic agents plant extracts, biopesticides, inorganiccrop protectants (such as copper), surfactants (such as rhamnolipids;Gandhi et al., 2007) or natural oils such as paraffinic oil and tea treeoil possessing pesticidal properties or chemical fungicides orbactericides with either single site, multisite or unknown mode ofaction. As defined herein, an “anti-phytopathogenic agent” is an agentwhich modulates the growth of a plant pathogen on a plant oralternatively prevents infection of a plant by a plant pathogen. A plantpathogen includes but is not limited to a fungus, bacteria, virus,insects, nematodes and/or mollusca.

In a particular embodiment, the anti-phytopathogenic agent is abiopesticide alternatively referred to as a biocontrol agent. Thisbiocontrol agent is in a more particular embodiment a non-Bacillus,non-Pseudomonas, non-Brevabacillus, non-Lecanicillium, non-Ampelomyces,non-Phoma, non-Pseudozyma biological control agent is an agent derivedfrom Streptomyces sp., Burkholderia sp., Trichoderma sp., Gliocladiumsp. Alternatively, the agent is a natural oil or oil-product havingfungicidal and/or insecticidal activity (e.g., paraffinic oil, tea treeoil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil).

As noted above, the anti-phytopathogenic agent may be a single siteanti-fungal agent which may include but is not limited to benzimidazole,a demethylation inhibitor (DMI) (e.g., imidazole, piperazine,pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine,phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide,carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatichydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin,acylamine, phthalimide, benzenoid (xylylalanine). In a more particularembodiment, the antifungal agent is a demethylation inhibitor selectedfrom the group consisting of imidazole (e.g., triflumizole), piperazine,pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole,propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole,fenbuconazole, hexaconazole, tebuconazole, tetraconazole,propiconazole). In a most particular embodiment, the antifungal agent ismyclobutanil. In yet another particular embodiment, the antifungal agentis a quinone outside inhibitor (e.g., strobulurin). The strobulurin mayinclude but is not limited to azoxystrobin, kresoxim-methoyl ortrifloxystrobin. In yet another particular embodiment, the anti-fungalagent is a quinone, e.g., quinoxyfen (5,7-dichloro-4-quinolyl4-fluorophenyl ether).

In yet a further embodiment, the antimicrobial agent is a multi-sitenon-inorganic, chemical fungicide selected from the group consisting ofa nitrile (e.g., chloronitrile or fludioxonil), quinoxaline, sulphamide,phosphonate, phosphite, dithiocarbamate, chloralkylhios,phenylpyridin-amine, cyano-acetamide oxime.

In yet another embodiment, the anti-phytopathogenic agent is ananti-bacterial agent. This anti-bacterial agent includes but is notlimited to carbamates, organophosphates, cyclodiene organochlorides,phenylpyrazoles, pyrethroids, pyrethrins, neonicotinoids,nitroguanadines, nicotine, Spinosyn, glycosides, juvenile hormoneanalogues and other insect growth regulators, pyridine azomethine,pyridine carboxamide, tetrazine, thiazolidinone, 2,4-diphenyloxzolinederivatives, organotin, pyrrole, buprofezin, hydramethylnon,naphtoquinon derivatives, pyridazinone, phenoxypyrazole, tetronic acid,carbazate, rotenone, organochlorine-diphenylaliphatics.

Uses

The said plant extract or formulated product can be used simultaneouslywith the other component or components in a tank mix or in a program(sequential application called rotation) with predetermined order andapplication interval during the growing season. When used in acombination with the above-mentioned pesticidal products, atconcentration lower than recommended in the product label, the combinedefficacy of the two or more products (one of which is the said plantextract) is in a preferred embodiment, higher than each individualcomponent's effect added together. Hence, the pesticidal effect isenhanced by synergism between these two (or more) products, and the riskfor the development of pesticide resistance among the plant pathogenicstrains is reduced.

Target plants to be protected within the scope of the present inventioncomprise, for example, the following species of plants: cereals (wheat,barley, rye, oats, rice, sorghum and related crops), beet (sugar beetand fodder beet), pommes and soft fruit (apples, pears, plum, peaches,almonds, cherries, strawberries, raspberries and blackberries),leguminous plants (beans, lentils, peas and soybeans), oil plants (rape,mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoabeans and ground nuts), cucurbits (cucumber, melons, pumpkins,eggplant), fiber plants (cotton, flax, hemp and jute), citrus fruit(oranges, lemon, grapefruits and mandarins), vegetables (spinach,lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes andpaprika), lauraceae (avocadoes, cinnamon and camphor) or plants such asmaze, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas andnatural rubber plants, as well as ornamentals (composites), areas ofgrass or general low cover crops which counteract erosion or desiccationof the soil and are useful in cultures of trees and perennials (fruitplantations, hop plantations, maze fields, vineyards, etc.).

The preferred method of applying combinations of products in the presentinvention is a foliar application (spraying, atomizing, dusting,scattering or pouring) with or without a carrier. The number ofapplications and the rate of application depend on the risk ofinfestation by a pathogen. For example, the foliar pesticidal treatmentswith combinations and mixtures covered in this patent can be made oncein every 7 to 14 days at 25 to 10,000-fold lower rates than recommendedin the product label. Product mixtures and combinations targeted in thepresent invention may also be applied to seeds by impregnating the seedseither with a liquid formulation containing the active ingredient orcoating them with a solid formulation. In special cases, further typesof application are also possible. These include soil drench or selectivetreatment of plant stems or buds.

The mixtures of the present invention and, where appropriate, a solid orliquid adjuvant are prepared in known manner. For example, the mixturesmay be prepared by homogeneously mixing and/or grinding the activeingredients with extenders such as solvents, solid carriers and, whereappropriate, surface-active compounds (surfactants). The compositionsmay also contain further ingredients such as stabilizers, viscosityregulators, binders, adjuvants as well as fertilizers or other activeingredients in order to obtain special effects.

EXAMPLES

As will be set forth below, in satisfaction of the foregoing objects andadvantages, examples of methods for increasing the efficacy of two ormore products by using them at rates that produce synergistic oradditive effects. The compositions and methods described here have beenproven effective in reducing the disease incidence and severity ingreenhouse-grown cucumbers (Cucumis sativus) but the concept can be usedeffectively for other plant varieties and species. The compositions andmethods are particularly effective against cucumber powdery mildew butthey can be applied to other fungal, bacterial, and viral plant diseasesas well such as grey mold, leaf spots, bacterial wilt, scab,anthracnose, tobacco mosaic virus etc.

Materials and Methods Powdery Mildew Examples I-V and X-XI

The studies were conducted in a green house. Experimental design of thestudies for synergism followed the Burpee and Latin (2008). Powderymildew, caused by Sphaerotheca fuliginea, was used for investigating theefficacy of the treatments.

Cucumber seeds cv. “SMR 58” (Irwin & Sons Ag Supply, Inc. Cheshire,Oreg.) were grown in plastic 4-inch pots with potting soil mix (RodMcLellan Company, Marysville, Ohio). The plants were treated at 2-trueleaf stage. The compounds at various rates were sprayed with a 2-oz mistsprayer at 2 ml per plant at upper side and 1 ml at lower side of theleaves. The treated plants were left for 3-4 hours under florescentlight to dry before inoculation.

A conidia suspension of Sphaerotheca fuliginea, the causal agent ofpowdery mildew disease on cucumber, was prepared by cutting diseasesleaves of the cucumber plants that are served for conservation ofinoculum. The suspension were adjusted to 2.0×10⁵ conidia per ml andapplied with a 2-oz mist sprayer at 2 ml per plant on the upper side ofthe leaves. The inoculated plants were placed in a greenhouse and thetreatments were arranged in randomized complete blocks with 4 to 6replicates at 25° C. to 30° C.

The disease severity (percentage area covered with colonies) of thefirst leaves were rated according to James (1971). Disease severity andpercentage control were analyzed with analysis of variance (ANOVA) andmeans of the treatments were compared with Fisher's Protected LeastDifference (LSD) at p=0.05 level. Synergetic effect was calculated andanalyzed with Limpel's formula (Limpel et al., 1962; Richer, 1987).

Lettuce Downy Mildew Examples VI-VII

Synergy between MBI-106 and acibenzolar-S-methyl or mefenoxam whichcontrol a specific class of pathogens Oomycetes was tested with lettucedowny mildew following Su et al. (2004).

To prepare inoculum of downy mildew, lettuce seeds were placed on 5-mmpetri-dishes with about 20-30 seeds in each and watered with sterilewater, then supplied with ½ strength of Hoagland's solution aftergermination. The petri dishes were placed at 20° C. growth chamber for7-10 days. The spores of Bremia lactucae was inoculated on lettucecotyledons and cultured for 7-10 days at 15° C. for sporulation tooccur. Cotyledons with sporulation were cut off and placed in a falcontube with sterile water. The cotyledons were vortexed for 15 secondsthree times and filtered through a 100-uμm mesh to collect sporangia.The solution was adjusted to 0.5 to 1.0×10⁵ spores/ml for inoculation.

Four test plants were seeded in each 2-in pots and then placed at 20° C.for 7-10 days to grow cotyledons. Plants were ready for test when thefirst true leaf was emerging.

The lettuce plants were treated with the materials and left to dry orovernight. Then the plants were inoculated with sporangial solution. Thetreatments were arranged in a randomized complete block design with fourreplicates. The inoculated plants were placed in darkness in growthchamber for 48 hours and then incubated under 12-h light period at 15°C. Eight to ten days after inoculation, the cotyledons were rated fordisease severity (percentage area covered with sporangiophores).

Seed Treatment Examples VIII-IX

The experiments described below were conducted with soybean but similarprocedures are used with other crops such as cereals, corn, cotton, andpotatoes.

The experiments were conducted in a greenhouse following the proceduredeveloped by Hwang et al. (2006). The isolate of Rhizoctonia solani wasgrown on potato dextrose agar plates and cultured for 5 days forinoculating oat or wheat grain. One liter of grain was soaked overnightat room temperature (25° C.) and rinsed three times with tape water. The8×12-in autoclave bags were filled with the grain up to ⅓ full wereautoclaved at 121° C. for 15 min. The autoclaved grain then was theninoculated with 5 pieces of 1×1 cm plug of the 5-day culture of R.solani per bag and cultured for 5 days at room temperature (25° C.). Thebags were left open in laminar flow hood to let the grain completelydry.

The blank seed sample of 10 g to be coated was placed in a flask.Coating agent SEPIRET® 1171-0 (Becker Underwood Ltd., Ames, Iowa) wasput into the flasks together with the compound of interest, and theflask was shaken vigorously so that the seed picked up the compound. Theprocedure was repeated with fresh seed sample of cvs. “White Lion”(Kitazawa Seed Co., Oakland, Calif.) or “Viking 2265” (Johnny's SelectedSeeds, Winslow, Me.) in the same flask for the treatments. After theseeds were coated, they were left to air dry before planting.

To prepare the soil, five hundred milliliters of the dried grains withinoculum were blended for 15 seconds three times and the powder wasmixed with sterile sand at 1:1 (v/v) to dilute the inoculum. The sandmix was further used to prepare soil mix at various proportions 1:350 to1:800 (inoculum:soil) for soybean to generate various disease levels inrepeated tests.

The coated seeds were planted in the pots with infested soil. There werethree replicates of each treatment which were arranged in a completerandomized block design and placed at 25 to 30° C. in a greenhouse.

After 10 to 20 days, depending on disease pressure and temperature,emergence of each treatment was rated and compared. Biomass was measuredby weighing the above-ground portion which was measured for eachreplicate of the plant material.

Example I Synergistic Effect Between Formulated R. sachalinensis Extractand Myclobutanil (Test I)

MBI-106 (formulated R. sachalinensis extract marketed as REGALIA® SC byMarrone Bio Innovations, Inc., Davis, Calif.) diluted at 10-fold lowerthan label rates of 1500× and 2000× and myclobutanil (formulated asRALLY® 40W, Dow AgroSciences LLC, Indianapolis, Ind.) at concentrationsof 0.25 ug/ml, 0.1 ug/ml, and 0.05 ug/ml (450 to 2.250-fold lower thanrecommended label rates) were applied alone or in tank mix.

Disease severity was significantly reduced in MBI-106 at 1500× and 2000×in combination with myclobutanil at 0.25 ug/ml than that when they wereused alone (Table 1). Significant increase in control efficacy was foundwith the combination of MBI-106 2000× tank mixed with myclobutanil atrates of 0.25 ug/ml and 0.05 ug/ml (Table 2).

Example II Synergistic Effect Between R. sachalinensis Extract andMyclobutanil (Test II)

In a second test using MBI-106 (formulated as REGALIA® SC) andmyclobutanil (formulated as RALLY® 40W), higher efficacy was found whenMBI-106 at 2000× (10-fold lower than label rate) dilution in combinationwith myclobutanil at rates of 0.25, 0.1, and 0.05 ug/ml, and whenMBI-106 at 1500× was tanked mixed with myclobutanil at 0.05 ug/ml (Table3). Significant synergistic effect was displayed when MBI-106 diluted at1500× and 2000× was combined with myclobutanil at the lowest rate of0.05 ug/ml (Table 4).

Example III Synergistic Effect Between formulated R. sachalinensisExtract and Quinoxyfen

MBI-106 diluted at 1500× or 2000× was applied alone or in combinationwith quinoxyfen (formulated as QUINTEC®, Dow AgroSciences LLC,Indianapolis, Ind.) at 0.05 and 0.01 ug/ml (2,000 to 10.000-fold lowerthan label rates) either alone or in a tank mix. The results are shownin Tables 5 and 6. The most significant synergistic effect and enhanceddisease control was found at R. sachalinensis product dilution of 2000×with quinoxyfen at 0.01 ug/mL. Quinoxyfen has a new multi-site mode ofaction affecting G-proteins in early cell signaling.

Example IV Synergistic Effect Between Formulated R. sachalinensisExtract and Azoxystrobin

MBI-106 diluted at 1500× or 2000× was applied alone or in combinationwith azoxystrobin (formulated as QUADRIS®, Syngenta Corporation,Wilmington, Del.) at a rate 0.25, 0.5, 1.0, 5.0, and 10 ug/mL (25 to1020-fold lower than recommended label rates) either alone or in a tankmix.

The results are shown in Tables 7 and 8. Of all tested combinations,mixes with R. sachalinensis extract at 2000× dilution and azoxystrobinat either 5.0 or 0.5 ug/mL provided the greatest synergy, and thefungicidal efficacy was significantly greater than expected compared tothe single-compound use data.

Example V Synergistic Effect Between Formulated R. sachalinensis Extractand Triflumizole

MBI-106 (formulated R. sachalinensis extract marketed as REGALIA® SC byMarrone Bio Innovations, Inc., Davis, Calif.) at 2500× dilution (10-foldlower than label rate) and triflumizole (formulated as PROCURE® 480SC,Chemtura Corporation, Middlebury, Conn.) at concentrations of 1.0 ug/ml,0.5 ug/ml, and 0.25 ug/ml (150 to 600-fold lower than recommended labelrates) were applied alone or in tank mix to.

Disease severity was significantly reduced (P<0.0001) in treatmentssprayed with MBI-106 at 2500× dilution in combination with triflumizolecompared with a treatment where MBI-106 was used alone (Table 9).Synergistic effect in efficacy was found at the combination of MBI-1062500× tank mixed with triflumizole at rates of 1.0 ug/ml, 0.5 ug/ml, and0.25 ug/ml (Table 10).

Example VI Synergistic Effect Between R. sachalinensis Extract andacibenzolar-S-methyl in Controlling Lettuce Downy Mildew

MBI-106 (formulated R. sachalinensis extract marketed as REGALIA® SC byMarrone Bio Innovations, Inc., Davis, Calif.) was used alone at 200×dilution or in combination with acibenzolar-S-methyl (formulated asACTIGARD®, Syngenta Crop Protection, Inc., Greensboro, N.C.) atconcentrations of 25 ug/ml to control lettuce downy mildew.

Disease severity was significantly reduced (p=0.0004) in MBI-106 at 200×dilution in combination with acibenzolar-S-methyl compared to treatmentsin which they were used alone (Table 11). There is a synergistic effectin efficacy in the tank mix of MBI-106 200× and acibenzolar-S-methyl(Table 12).

Example VII Synergistic Effect Between R. sachalinensis Extract andMefenoxam in Controlling Lettuce Downy Mildew

To investigate the synergistic effect of Reynoutria and MBI-106,formulated product REGALIA® ME by Marrone Bio Innovations, Inc., Davis,Calif. was used at 400× dilution rate in combination with mefenoxam(formulated as RIDOMIL GOLD®, Syngenta Crop Protection, Inc.,Greensboro, N.C.) at concentrations of 37.5 μg/ml, 75.0 μg/ml, and 150μg/ml to control lettuce downy mildew.

Disease severity was significantly reduced (p<0.0001) in MBI-106 at 400×dilution in combination with mefenoxam compared to treatments in whichthey were used alone at each concentration (Table 13). Synergisticeffect in efficacy of the tank mix of MBI-106 200× and mefenoxam wasfound in combinations of MBI-106 and each concentration of mefenoxam(Table 14).

Example VIII R. sachalinensis Extract as a Seed Treatment Agent and itsSynergy with Azoxystrobin in Controlling Rhizoctonia solani

Reynoutria sachalinensis was extracted with ethanol at 5% (w/w) and usedfor seed coating at 0.2117 g/kg seed, either alone or in combinationwith azoxystrobin (QUADRIS®, Syngenta Crop Protection, Inc., Greensboro,N.C.) at 0.0298 g/kg seed to control Rhizoctonia solani on soybean. Theemergence rate was higher in the inoculated seeds treated with MBI-106compared to the inoculated untreated control, and when used incombination with azoxystrobin, the emergence rate was higher than wheneither product was used alone (Table 15). A synergistic effect was foundwhen both materials were used in combination (Table 16).

Example IX R. sachalinensis Extract as a Seed Treatment Agent andSynergy with Fludioxonil in Controlling Rhizoctonia solani

The ethanol extract of R. sachalinensis was also used at the rates of0.03175 g/kg seed and 0.635 g/kg seed for seed coating, either alone orin combination with fludioxonil (formulated as Scholar® by Syngenta CropProtection, Inc., Greensboro, N.C.) to control Rhizoctonia solani onsoybean. The emergence rates and biomass were higher in the MBI-106treated seeds compared to the inoculated untreated control and alsohigher when used in combination with fludioxonil at the rate of 0.0596g/kg seed (Table 17). Synergistic effect was found in the two rates ofMBI-106 when used in combination with fludioxonil (Table 18).

Example X Synergistic Effect Between Formulated R. sachalinensis Extractand Propiconazole in Controlling Cucumber Powdery Mildew

MBI-106 (formulated R. sachalinensis extract marketed as REGALIA® ME byMarrone Bio Innovations, Inc., Davis, Calif.) at 2500× dilution andpropiconazole (formulated as PROPIMAX® EC, Dow AgroSciences LLC,Indianapolis, Ind.) at concentrations of 1.0 ug/ml were applied alone orin tank mix.

Disease severity was significantly reduced (P<0.0001) in treatmentssprayed with MBI-106 at 2500× dilution in combination with propiconazole(Table 19). There is a synergistic effect in control efficacy in thecombination of MBI-106 2500× tank mixed with propiconazole (Table 20).

Example XI Synergistic Effect Between R. sachalinensis Extract andQuinoxifen (Test II) in Controlling Cucumber Powdery Mildew

MBI-106 (formulated R. sachalinensis extract marketed as REGALIA® SC byMarrone Bio Innovations, Inc., Davis, Calif.) was used alone at 2000×dilution or in combination with azoxystrobin at three concentration 0.5,0.25, and 0.1 ug/ml to control cucumber powdery mildew.

The control efficacy in MBI-106 at 2000× dilution in combination withazoxystrobin treatments are higher that they are used alone (Table 21).Synergistic effect in control efficacy of the tank mix of MBI-106 2000×exists in combinations of MBI-106 and each concentration of azoxystrobin(Table 22).

Tables

TABLE 1 Disease severity and percentage control of MBI-106 andmyclobutanil (RALLY ® 40W) when used alone or in tank mix.¹ TreatmentDilution/Rate Severity (%)^(z) Control (%) Untreated control N/A 98.3 a0.0 e myclobutanil 0.25 ug/ml 16.7 cd 83.1 bc myclobutanil  0.1 ug/ml95.0 a 3.2 e myclobutanil 0.05 ug/ml 95.8 a 2.5 e Reynoutria extract1500× 15.8 cd 84.0 bc Reynoutria extract 2000× 35.8 b 63.5 d Reynoutriaextract + 1500× 1.2 e 98.8 a myclobutanil 0.25 ug/ml Reynoutriaextract + 2000× 0.2 e 99.8 a myclobutanil 0.25 ug/ml Reynoutriaextract + 1500× 11.8 de 87.9 ab myclobutanil  0.1 ug/ml Reynoutriaextract + 2000× 27.5 bc 72.2 dc myclobutanil  0.1 ug/ml Reynoutriaextract + 1500× 17.5 cd 82.4 bc myclobutanil 0.05 ug/ml Reynoutriaextract + 2000× 20.8 cd 79.0 bc myclobutanil 0.05 ug/ml P < 0.0001 P <0.0001 ¹Data in Column 3 (Severity (%)) are means of six replicates.Means with the same letter in a column are not significantly differentaccording to Fisher's Protected Least Significant Difference (LSD) at P= 0.05 level.

TABLE 2 Expected efficacy (Ee)² of each product combination, and thestatistical significance of detected synergism between MBI-106(REGALIA ®SC) and myclobutanil (RALLY ® 40W) Treatment Dilution/RateControl (%) E_(e) ³ T-test Reynoutria extract + 1500× 98.8 97.3 n.s.myclobutanil 0.25 ug/ml Reynoutria extract + 2000× 99.8 93.8 ***myclobutanil 0.25 ug/ml Reynoutria extract + 1500× 87.9 84.5 n.s.myclobutanil  0.1 ug/ml Reynoutria extract + 2000× 72.2 64.7 n.s.⁴myclobutanil  0.1 ug/ml Reynoutria extract + 1500× 82.4 84.4 n.s.myclobutanil 0.05 ug/ml Reynoutria extract + 2000× 79.0 64.4 *⁵myclobutanil 0.05 ug/ml ²E_(e) is the Expected efficacy and isdetermined with the Limpel's formula E_(e) = X + Y − (XY)/100 (Limpel etal., 1962; Richer, 1987). ³Data are means of six replicates. Means withthe same letter in a column are not significantly different according toFisher's Protected Least Significant Difference (LSD) at P = 0.05 level.⁴n.s.: Not significant ⁵* and *** Significant at P < 0.05 and 0.001respectively.

TABLE 3 Disease severity and percentage control of MBI-106 (REGALIA ®SC) and myclobutanil (RALLY ® 40W) when used alone or in tank mix in arepeated test. Treatment Dilution/Rate Severity (%)^(z6) Control (%)Untreated control N/A 91.3 a 0.0 e myclobutanil 0.25 ug/ml 53.8 bcd 41.3dc myclobutanil  0.1 ug/ml 80.0 ab 12.2 de myclobutanil 0.05 ug/ml 93.8a −2.8 e Reynoutria extract 1500× 20.0 ef 77.9 ab Reynoutria extract2000× 63.8 abc 29.8 cde Reynoutria extract + 1500× 46.3 cde 48.8 bcmyclobutanil 0.25 ug/ml Reynoutria extract + 2000× 33.8 cdef 62.7 bcmyclobutanil 0.25 ug/ml Reynoutria extract + 1500× 33.8 cdef 62.6 bcmyclobutanil  0.1 ug/ml Reynoutria extract + 2000× 38.8 cde 57.3 bcmyclobutanil  0.1 ug/ml Reynoutria extract + 1500× 1.8 f 98.1 amyclobutanil 0.05 ug/ml Reynoutria extract + 2000× 21.3 def 77.0 abmyclobutanil 0.05 ug/ml P < 0.0001 P < 0.0001 ⁶Data are means of fourreplicates. Means with the same letter in a column are not significantlydifferent according to Fisher's Protected Least Significant Difference(LSD) at P = 0.05 level.

TABLE 4 Synergistic effect between MBI-106 (REGALIA ® SC) andmyclobutanil (RALLY ® 40W) in a repeated test. Treatment Dilution/RateControl (%) E_(e) ^(z) T test Reynoutria extract + 1500× myclobutanil0.25 ug/ml 48.8 87.0 n.s. Reynoutria extract + 2000× myclobutanil 0.25ug/ml 62.7 58.8 n.s. Reynoutria extract + 1500× myclobutanil  0.1 ug/ml62.6 80.6 n.s. Reynoutria extract + 2000× myclobutanil  0.1 ug/ml 57.338.3 n.s. Reynoutria extract + 1500× myclobutanil 0.05 ug/ml 98.1 77.3*** Reynoutria extract + 2000× myclobutanil 0.05 ug/ml 77.0 27.8 **^(z)E_(e) is the Expected efficacy and is determined with the Limpel'sformula E_(e) = X + Y − (XY)/100 (Limpel et al., 1962; Richer, 1987). **and *** Significant at P < 0.01 and 0.001 respectively. n.s.: Notsignificant

TABLE 5 Disease severity and percentage control of MBI-106 (formulatedR. sachalinensis extract) and quinoxyfen (QUINTEC ®) when used alone orin tank mix. Treatment Dilution/Rate Severity (%) Control (%) Untreatedcontrol N/A 91.3 a 0 Reynoutria extract 1500× 20.0 c 77.93 Reynoutriaextract 2000× 63.8 b 29.75 quinoxyfen 0.05 ug/ml 30.0 c 66.95 quinoxyfen0.01 ug/ml 90.0 a 1.33 Reynoutria extract + 1500× quinoxyfen 0.05 ug/ml25.0 c 72.65 Reynoutria extract + 2000× quinoxyfen 0.05 ug/ml 12.5 c86.25 Reynoutria extract + 1500× quinoxyfen 0.01 ug/ml 22.5 c 75.28Reynoutria extract + 2000× quinoxyfen 0.01 ug/ml 13.8 c 85.1 n = 4, LSDP < 0.0001 P < 0.0001 Data are means of four replicates. Means with thesame letter in a column are not significantly different according toFisher's Protected Least Significant Difference (LSD) at P = 0.05 level.

TABLE 6 Synergistic effect between MBI-106 (Reynoutria extract,REGALIA ® SC) and quinoxyfen (QUINTEC ®) Treatment Dilution/Rate Control(%) E_(e) T-test Reynoutria extract + 1500× 72.65 92.7 * quinoxyfen 0.05ug/ml Reynoutria extract + 2000× 86.25 76.8 n.s. quinoxyfen 0.05 ug/mlReynoutria extract + 1500× 75.28 78.2 n.s. quinoxyfen 0.01 ug/mlReynoutria extract + 2000× 85.1 30.7 ** quinoxyfen 0.01 ug/ml ^(Z)E_(e)is the Expected efficacy and is determined with the Limpel's formulaE_(e) = X + Y − (XY)/100 (Limpel et al., 1962; Richer, 1987). * and **Significant at P < 0.05 and 0.01 respectively. n.s.: Not significant.

TABLE 7 Disease severity and percentage control of powdery mildew usingMBI-106 (R. sachalinensis extract, REGALIA ® SC) and azoxystrobin(QUADRIS ®). Severity Control Treatment Dilution/Rate (%) (%) Untreatedcontrol N/A 92.5 a 0.0 h azoxystrobin 10.0 ug/ml  11.3 efg 87.6 abcazoxystrobin 5.0 ug/ml 5.3 fg 94.3 ab azoxystrobin 1.0 ug/ml 30.0 cdef68.1 bcdef azoxystrobin 0.5 ug/ml 48.8 bc 48.0 fg azoxystrobin 0.25ug/ml  45.0 bcd 51.8 efg Reynoutria extract 1500x 11.3 efg 88.0 abcReynoutria extract 2000x 61.3 b 34.3 g Reynoutria extract 2500x 48.8 bc47.0 fg Reynoutria extract + 1500x 3.0 fg 96.8 ab azoxystrobin 10.0ug/ml  Reynoutria extract + 2000x 4.0 fg 95.6 ab azoxystrobin 10.0ug/ml  Reynoutria extract + 2500x 1.8 fg 98.1 ab azoxystrobin 10.0ug/ml  Reynoutria extract + 1500x 1.0 g 98.9 ab azoxystrobin 5.0 ug/mlReynoutria extract + 2000x 0.8 g 99.2 a azoxystrobin 5.0 ug/mlReynoutria extract + 2500x 2.0 fg 97.9 ab azoxystrobin 5.0 ug/mlReynoutria extract + 1500x 11.3 efg 87.6 abc azoxystrobin 1.0 ug/mlReynoutria extract + 2000x 12.5 efg 86.3 abcd azoxystrobin 1.0 ug/mlReynoutria extract + 2500x 25.0 cdefg 73.4 abcdef azoxystrobin 1.0 ug/mlReynoutria extract + 1500x 16.3 defg 82.2 abcde azoxystrobin 0.5 ug/mlReynoutria extract + 2000x 11.3 efg 87.7 abc azoxystrobin 0.5 ug/mlReynoutria extract + 2500x 43.8 bcd 52.1 efg azoxystrobin 0.5 ug/mlReynoutria extract + 1500x 40.0 bcde 56.4 defg azoxystrobin 0.25 ug/ml Reynoutria extract + 2000x 25.0 cdefg 73.0 abcdef azoxystrobin 0.25ug/ml  Reynoutria extract + 2500x 36.3 bcde 60.5 cdefg azoxystrobin 0.25ug/ml  N = 4 P < 0.0001 P < 0.0001

TABLE 8 Synergistic effect between MBI-106 and azoxystrobin. Onlytreatments with statistically significant synergistic effect areincluded. Treatment Rate/Dilution Control (%) E_(e) T-test Reynoutriaextract + 2000× 95.6 91.9 * azoxystrobin 10.0 ug/ml Reynoutria extract +2500× 98.1 93.4 * azoxystrobin 10.0 ug/ml Reynoutria extract + 2000×99.2 96.3 ** azoxystrobin 5.0 ug/ml Reynoutria extract + 2000× 87.7 65.8** azoxystrobin 0.5 ug/ml

TABLE 9 Disease severity and percentage control of R. sachalinensis andtriflumizole (PROCURE ® 480SC) when used alone or in tank mix. TreatmentDilution/Rate Severity (%) Control (%) Untreated control N/A 97.5 a 0.0Reynoutria extract 2500× 23.8 b 75.6 triflumizole 1.0 ug/ml 77.5 a 20.5triflumizole 0.5 ug/ml 86.3 a 11.5 triflumizole 0.25 ug/ml  95.0 a 2.6Reynoutria extract + 2500× 6.3 b 93.6 triflumizole 1.0 ug/ml Reynoutriaextract + 2500× 15.0 b 84.6 triflumizole 0.5 ug/ml Reynoutria extract +2500× 11.5 b 88.2 triflumizole 0.25 ug/ml  Data in Column 3 (Severity(%)) are means of four replicates. Means with the same letter in acolumn are not significantly different according to Fisher's ProtectedLeast Significant Difference (LSD) at P = 0.05 level.

TABLE 10 The expected efficacy (E_(e)) of each product combination,control efficacy (E), and the synergistic effect (E/E_(e) > 1.0) betweenMBI-106 (REGALIA ® SC) and triflumizole (PROCURE ® 480SC) Tank mixDilution/Rate Control (%) E_(e) E/E_(e) Reynoutria extract + 2500× 93.680.6 1.2 triflumizole 1.0 ug/ml Reynoutria extract + 2500× 84.6 78.4 1.1triflumizole 0.5 ug/ml Reynoutria extract + 2500× 88.2 76.2 1.2triflumizole 0.25 ug/ml  E_(e) is the expected efficacy and isdetermined with the Limpel's formula E_(e) = X + Y − (XY)/100 (Limpel etal., 1962; Richer, 1987).

TABLE 11 Disease severity and percentage control of MBI- 106 (REGALIA ®SC) and acibenzolar-S- methyl (ACTIGARD ®) when used alone or in tankmix. Treatment Dilution/Rate Severity (%) Control (%) Untreated controlN/A 90.0 a 0.0 acibenzolar-S-methyl 25 ug/ml 68.9 ab 23.4 Reynoutriaextract 200× 57.0 b 36.6 Reynoutria extract + 200× 14.2 c 84.2acibenzolar-S-methyl 25 ug/ml

TABLE 12 The expected efficacy (E_(e)) of each product combination,control efficacy (E), and the synergistic effect (E/E_(e) > 1.0) betweenMBI-106 (REGALIA ® SC) and acibenzolar-S-methyl (ACTIGARD ®). Tank mixControl (%) E_(e) E/E_(e) Reynoutria extract + 84.2 51.5 1.6acibenzolar-S-methyl

TABLE 13 Disease severity and percentage control of MBI-106 (REGALIA ®ME) and mefenoxam(RIDOMIL GOLD ® SL) Treatment Dilution/Rate Severity(%) Control (%) Untreated control N/A 87.0 ab 0.0 mefenoxam 150 ug/ml44.8 de 48.5 mefenoxam  75 ug/ml 54.2 cd 37.7 mefenoxam 37.5 ug/ml  89.3a −2.6 Reynoutria extract 400× 68.0 bc 21.8 Reynoutria extract + 400×14.5 f 83.3 mefenoxam 150 ug/ml Reynoutria extract + 400× 28.6 ef 67.1mefenoxam  75 ug/ml Reynoutria extract + 400× 59.2 cd 32.0 mefenoxam37.5 ug/ml 

TABLE 14 The expected efficacy (E_(e)) of each product combination,actual control efficacy (E), and detected synergism (E/E_(e) > 1.0)between MBI-106 (REGALIA ® ME) and mefenoxam (RIDOMIL GOLD ® SL). Tankmix Dilution/Rate Control (%) E_(e) E/E_(e) Reynoutria extract + 400×83.3 59.7 1.4 mefenoxam 150 ug/ml Reynoutria extract + 400× 67.1 51.31.3 mefenoxam  75 ug/ml Reynoutria extract + 400× 32.0 19.7 1.6mefenoxam 37.5 ug/ml 

TABLE 15 Emergence of soybean seedling and control of damping off byMBI- 106 (ethanol extract) and azoxystrobin (QUADRIS ®). Treatment RateEmergence (%) Control (%) Non-inoculated control N/A 90.1 a N/AInoculated control N/A 4.9 c 0.0 Inoculated Reynoutria 0.02117 g/kg 11.1 c 6.2 extract Inoculated Reynoutria 0.02117 g/kg  92.6 a 87.7extract + azoxystrobin 0.0298 g/kg Inoculated azoxystrobin 0.0298 g/kg86.4 a 81.5

TABLE 16 The expected efficacy (E_(e)) of each product combination,actual control efficacy (E), and detected synergism (E/E_(e) > 1.0)between MBI-106 (ethanol extract) and azoxystrobin (QUADRIS ®). Tank mixRate Control (%) E_(e) E/E_(e) Reynoutria extract + 0.02117 g/kg 87.782.6 1.1 azoxystrobin  0.0298 g/kg

TABLE 17 Emergence of soybean seedlings and control of damping off byMBI-106 (Reynoutria ethanol extract) and fludioxonil (SCHOLAR ®).Emergence Bio mass Treatment Rate (%) (g) Control (%) Inoculated controlN/A 7.4 c 0.8 c 0.0 Inoculated Reynoutria extract 0.635 g/kg 9.9 c 1.4 c2.5 Inoculated Reynoutria extract 0.03175 g/kg 16.0 c 2.9 c 8.6Inoculated Reynoutria extract + 0.635 g/kg 61.8 a 17.0 a 54.4fludioxonil 0.0596 g ai/kg Inoculated Reynoutria extract + 0.03175 g/kg50.6 a 12.5 b 43.2 fludioxonil 0.0596 g ai/kg Inoculated fludioxonil0.0596 g ai/kg 35.8 b 8.7 b 28.4

TABLE 18 The expected efficacy (E_(e)) of each product combination,actual control efficacy (E), and detected synergism (E/E_(e) > 1.0)between MBI-106 (ethanol extract) and fludioxonil (SCHOLAR ®). Tank mixRate Control (%) E_(e) E/E_(e) Inoculated Reynoutria 0.635 g/kg 54.430.2 1.8 extract + fludioxonil 0.0596 g ai/kg Inoculated Reynoutria0.03175 g/kg 43.2 34.6 1.2 extract + fludioxonil 0.0596 g ai/kg

TABLE 19 Disease severity and percentage control of R. sachalinensis andpropiconazole (PROPIMAX ®) when used alone or in tank mix in controllingcucumber powdery mildew. Treatment Dilution/Rate Severity (%) Control(%) Untreated control N/A 98.8 a 0.0 propiconazole 1.0 ug/ml 97.5 a 1.3Reynoutria extract 2500× 61.3 b 38.0 Reynoutria + 2500× 51.3 c 48.1propiconazole 1.0 ug/ml

TABLE 20 The expected efficacy (E_(e)) of each product combination,control efficacy (E), and the synergistic effect (E/E_(e) > 1.0) betweenMBI-106 (REGALIA ® ME) and propiconazole (PROPIMAX ®). Tank mixDilution/Rate control (%) E^(e) E/e_(e) Reynoutria extract + 2500× 48.138.8 1.2 propiconazole 1.0 ug/ml

TABLE 21 Disease severity and percentage control of R. sachalinensis andazoxystrobin (QUADRIS ®) when used alone or in tank mix in controllingcucumber powdery mildew (

  Treatment Dilution/Rate Severity % Control (%) Untreated control N/A80.0 a 13.5 azoxystrobin 0.5 ug/ml 35.0 cd 62.2 azoxystrobin 0.25 ug/ml 80.0 a 13.5 azoxystrobin 0.1 ug/ml 72.5 abc 21.6 Reynoutria extract2000× 75.0 ab 18.9 Reynoutria extract + 2000× 11.3 d 87.8 azoxystrobin0.5 ug/ml Reynoutria extract + 2000× 41.3 abcd 55.4 azoxystrobin 0.25ug/ml  Reynoutria extract + 2000× 36.5 bcd 60.5 azoxystrobin 0.1 ug/ml

indicates data missing or illegible when filed

TABLE 22 The expected efficacy (E_(e)) of each product combination,control efficacy (E), and the synergistic effect (E/E_(e) > 1.0) betweenMBI-106 (Regalia ® SC) and azoxystrobin (Quadris ®) (Test II). Tank mixDilution/Rate Control (%) E_(e) E/E_(e) Reynoutria extract + 2000× 87.869.3 1.3 azoxystrobin 0.5 ug/ml Reynoutria extract + 2000× 55.4 29.9 1.9azoxystrobin 0.25 ug/ml  Reynoutria extract + 2000× 60.5 36.4 1.7azoxystrobin 0.1 ug/ml

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1. A combination comprising: (a) an extract containing one or moreanthraquinone derivatives which induce plant resistance tophytopathogens and derived from a plant and (b) one or moreanti-phytopathogenic agents selected from the group consisting of: (i) anon-benzodiathiazole, non-Vitamin E, non-organophosphorus anti-microbialagent, which lacks non-elemental or non-wettable, sulfur, (ii) asurfactant having fungicidal activity, and/or (iii) a non-Bacillus,non-Pseudomonas, non-Brevabacillus, non-Lecanicillium, non-Ampelomyces,non-Phoma, non-Pseudozyma biological control agent.
 2. The combinationaccording to claim 1 wherein said anti-microbial agent is a chemicalfungicide, bactericide, insecticide or nematicide.
 3. The combinationaccording to claim 1, wherein said non-Bacillus, non-Pseudomonas,non-Brevabacillus, non-Lecanicillium, non-Ampelomyces, non-Phoma,non-Pseudozyma biological control agent is selected from the groupconsisting of an agent derived from Streptomyces sp., Burkholderia sp.,Trichoderma sp., Gliocladium sp. and a natural oil or oil-product havingfungicidal and/or insecticidal activity.
 4. The combination according toclaim 1, wherein said extract is at least one of: (a) derived from thefamily Polygonaceae (b) contains an anthraquinone derivative containingat least physcion, (c) derived from a Reynoutria or Rheum species. 5.The combination according to claims 1, wherein said anti-microbialagent, is selected from the group consisting of (a) a single siteanti-fungal agent, wherein said single-site anti-fungal agent isselected from the group consisting of benzimidazole, morpholine,hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outsideinhibitor, quinoline, dicarboximide, carboximide, phenylamide,anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid,hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid(xylylalanine); (b) a demethylation inhibitor selected from the groupconsisting of imidazole, piperazine, pyrimidine, and triazole, (c) anatural oil or oil-product having fungicidal and/or insecticidalactivity and (c) a multi-site non-inorganic, chemical fungicide selectedfrom the group consisting of a nitrile, copper, quinoxaline, sulphamide,phosphonate, phosphite, dithiocarbamate, chloralkylhios,phenylpyridin-amine, cyano-acetamide oxime, fludioxonil and mefenoxam.6. The combination according to claim 1, wherein said antimicrobialagent is a (a) triazole, wherein said triazole is selected from thegroup consisting of bitertanol, myclobutanil, penconazole,propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole,fenbuconazole, hexaconazole, tebuconazole, tetraconazole, (b) animidazole, wherein said imidazole is fenamidone, fenapanil, iprodione,triflumizole (c) a nitrile, wherein said nitrile is chloronitrile andfludioxonil; (c) a stroliburin; (d) quinone, wherein said quinone isquinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether).
 7. Thecombination according to claim 1, wherein said antimicrobial agent isstrobilurin, wherein said strobilurin is azoxystrobin, kresoxim-methoylor trifloxystrobin.
 8. The combination according to claim 1, whereinsaid combination is a composition.
 9. A synergistic combination for usein modulating phytopathogenic infection comprising (a) an extractderived from a plant, wherein said plant contains anthraquinonederivatives that induce plant resistance to phytopathogens and (b) anon-Vitamin E, non-organophosphorus anti-microbial agent, which lacksnon-elemental or non-wettable, sulfur.
 10. The combination according toclaim 9, wherein said combination is a composition.
 11. A method formodulating phytopathogenic infection in a plant comprising applying tothe plant and/or seeds thereof and/or substrate used for growing saidplant an amount of the combination of claim 1 effective to modulate saidphytopathogenic infection.
 12. A method for modulating phytopathogenicinfection in a plant comprising applying to the plant and/or seedsthereof and/or substrate used for growing said plant an amount of thecombination of claim 9 effective to modulate said phytopathogenicinfection.
 13. A method for decreasing the emergence of resistance of aphytopathogen to an anti-phytopathogenic agent selected from the groupconsisting of: (i) a non-Vitamin E, non-organophosphorus anti-microbialagent, which lacks non-elemental or non-wettable sulfur (ii) asurfactant having fungicidal activity and (iii) a non-Bacillus,non-Pseudomonas, non-Brevabacillus, non-Lecanicillium, non-Ampelomyces,non-Phoma, non-Pseudozyma biological control agent comprising applyingto a plant in need thereof an amount of the combination of claim 1effective for decreasing said resistance of said phytopathogen to anantiphytopathogenic agent.
 13. A method for decreasing the emergence ofresistance of a phytopathogen to an anti-phytopathogenic agent selectedfrom the group consisting of: (i) a non-Vitamin E, non-organophosphorusanti-microbial agent, which lacks non-elemental or non-wettable sulfur(ii) a surfactant having fungicidal activity and (iii) a non-Bacillus,non-Pseudomonas, non-Brevabacillus, non-Lecanicillium, non-Ampelomyces,non-Phoma, non-Pseudozyma biological control agent comprising applyingto a plant in need thereof an amount of the combination of claim 9effective for decreasing said resistance of said phytopathogen to anantiphytopathogenic agent.
 14. A kit comprising (a) an extract derivedfrom the family Polygonaceae and (b) one or more anti-phytopathogenicagents selected from the group consisting of: (i) non-elemental ornon-wettable, sulfur, non-benzodiathiazole, non-Vitamin E,non-organophosphorus, anti-microbial agent, (ii) a surfactant havingfungicidal activity and (iii) a non-Bacillus, non-Pseudomonas,non-Brevabacillus, non-Lecanicillium, non-Ampelomyces, non-Phoma,non-Pseudozyma biological control agent.